Time-lapse imaging of molecular evolution by high-throughput sequencing

Abstract

High-throughput sequencing of in vitro selection could artificially provide large quantities of relic sequences from known times of molecular evolution. Here, we demonstrate how it can be used to reconstruct an empirical genealogical evolutionary (EGE) tree of an aptamer family. In contrast to classical phylogenetic trees, this tree-diagram represents proliferation and extinction of sequences within a population during rounds of selection. Such information, which corresponds to their evolutionary fitness, is used to infer which sequences may have been mutated through the selection process that led to the appearance and spreading of new sequences. This approach was validated by the re-analysis of an in vitro selection that had previously identified an aptamer against Annexin A2. It revealed that this aptamer might be the descendant of a sequence that was more highly amplified in early rounds. It also succeeded in predicting improved variants of this aptamer and providing a means to understand the influence of selection pressure on evolution. This is the first demonstration that HTS can provide time-lapse imaging of the evolutionary pathway that is taken by a macromolecule during in vitro selection to evolve by successive mutations through better fitness.

µIVC-Useq: a microfluidic-assisted high-throughput functional screening in tandem with next-generation sequencing and artificial neural network to rapidly characterize RNA molecules

“…However, technological advances like NGS now allow the whole selection process to be characterized at once and is commonly used to assist hits identification ( Nguyen Quang et al 2016 ; Kinghorn et al 2017 ; Yokobayashi 2019 ). The global view offered by such analyses makes it possible to rapidly identify variants of interest, even though they are underrepresented at the end of the selection process ( Nguyen Quang et al 2018 ; Autour et al 2019 ). Yet, these approaches may still require significant manual analyses and may be fastidious.…”

FASTAptameR 2.0: A web tool for combinatorial sequence selections

“…Under optimal circumstances, the evolutionary dynamics of populations undergoing selection reflect the relative fitness of each species, with high-fitness sequences typically enriching during selection and low-fitness sequences depleting. Thus, common analytic tasks of any combinatorial selection include counting the number of occurrences for each sequence, 24 , 25 calculating enrichment of sequences between two or more rounds, 25 , 26 , 27 filtering sequences based on the number of reads present in one or multiple rounds, 28 clustering related sequences, 25 , 29 , 30 , 31 and in some cases analyzing predicted structure motifs. 29 , 32 , 33 , 34 , 35 , 36 High-throughput sequencing (HTS) provides large volumes of data for these analyses and can yield high-resolution insights.…”

“…Thus, common analytic tasks of any combinatorial selection include counting the number of occurrences for each sequence, 24 , 25 calculating enrichment of sequences between two or more rounds, 25 , 26 , 27 filtering sequences based on the number of reads present in one or multiple rounds, 28 clustering related sequences, 25 , 29 , 30 , 31 and in some cases analyzing predicted structure motifs. 29 , 32 , 33 , 34 , 35 , 36 High-throughput sequencing (HTS) provides large volumes of data for these analyses and can yield high-resolution insights. Many specialized bioinformatics toolkits have been developed to enable this analytical workflow, 37 and several of these tools include graphical user interfaces to visualize HTS data during the analysis.…”

“… 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 HTS investigation of in vitro selection pools has revealed the distribution and relative frequencies of individual sequences and groups of related sequences as the populations evolve through the course of the experiment. 48 , 49 Such data can inform on the success of the selection, 45 , 50 aptamer-target interactions, 51 , 52 , 53 , 54 the mutation and fitness landscape, 29 , 44 , 55 structure-function relations, biological constraints, and more.…”

EasyDIVER: A Pipeline for Assembling and Counting High-Throughput Sequencing Data from In Vitro Evolution of Nucleic Acids or Peptides

“…In vitro evolution is a widely used method to isolate functional sequences with desired properties. These experiments, particularly RNA and DNA selections, are often analyzed by High-Throughput Sequencing (HTS) on the Illumina platform (Yokobayashi 2019;Blanco et al 2019;Nguyen Quang et al 2018). Increasingly, HTS analysis is being applied to peptide or protein selections, such as mRNA display.…”